TY - JOUR

T1 - Crosslinking of Dam methyltransferase with S-adenosyl-methionine

AU - Wenzel, C

AU - Moulard, M

AU - Løbner-Olesen, A

AU - Guschlbauer, W

PY - 1991/3/11

Y1 - 1991/3/11

N2 - Highly purified DNA-adenine methyltransferase was irradiated in the presence of different concentrations of radiolabelled S-adenosyl-methionine (AdoMet) with a conventional Mineralight UV-lamp from several minutes up to 1 h while incubating in ice. Incorporation of radioactivity was monitored by electrophoresis of the crosslink between S-adenosyl-methionine and Dam methylase on SDS-polyacrylamide gels followed by fluorography. Crosslinking reached a maximum in presence of 10 microM S-adenosyl-methionine; it was inhibited in the presence of substances which competitively inhibit methylation of DNA by Dam methylase, like sinefungin or S-adenosyl-homocysteine, but not in the presence of non-inhibitors like ATP or S-isobutyl-adenosine. The crosslink obtained was resistant against a wide range of even drastic conditions commonly used in protein and peptide chemistry. Proteins, which do not bind S-adenosyl-methionine, as well as heat inactivated Dam methylase were not photolabelled. After limited proteolysis the radioactive label appeared only in certain of the peptides obtained. From Western blots carried out with polyclonal antibodies produced against a synthetic peptide corresponding in its sequence to amino acids 92-106 of the Dam methylase, the crosslinking of AdoMet could be tentatively mapped at a position after amino acid 106.

AB - Highly purified DNA-adenine methyltransferase was irradiated in the presence of different concentrations of radiolabelled S-adenosyl-methionine (AdoMet) with a conventional Mineralight UV-lamp from several minutes up to 1 h while incubating in ice. Incorporation of radioactivity was monitored by electrophoresis of the crosslink between S-adenosyl-methionine and Dam methylase on SDS-polyacrylamide gels followed by fluorography. Crosslinking reached a maximum in presence of 10 microM S-adenosyl-methionine; it was inhibited in the presence of substances which competitively inhibit methylation of DNA by Dam methylase, like sinefungin or S-adenosyl-homocysteine, but not in the presence of non-inhibitors like ATP or S-isobutyl-adenosine. The crosslink obtained was resistant against a wide range of even drastic conditions commonly used in protein and peptide chemistry. Proteins, which do not bind S-adenosyl-methionine, as well as heat inactivated Dam methylase were not photolabelled. After limited proteolysis the radioactive label appeared only in certain of the peptides obtained. From Western blots carried out with polyclonal antibodies produced against a synthetic peptide corresponding in its sequence to amino acids 92-106 of the Dam methylase, the crosslinking of AdoMet could be tentatively mapped at a position after amino acid 106.

KW - Binding, Competitive

KW - Cross-Linking Reagents/metabolism

KW - Endopeptidases/pharmacology

KW - Enzyme Activation

KW - Enzyme Stability/drug effects

KW - Hot Temperature

KW - Methyltransferases/metabolism

KW - S-Adenosylmethionine/metabolism

KW - Site-Specific DNA-Methyltransferase (Adenine-Specific)

KW - Substrate Specificity/drug effects

KW - Ultraviolet Rays

M3 - Journal article

C2 - 2009958

VL - 280

SP - 147

EP - 151

JO - F E B S Letters

JF - F E B S Letters

SN - 0014-5793

IS - 1

ER -